The long term goal of this project is to understand how diabetes affects the heart and derive better treatment options. This is especially important given the high incidence of diabetes and ensuing cardiovascular complications. Indeed, diabetes can induce changes to cardiac function in the absence of other risk factors through mechanisms that are not completely clear. The focus of this proposal is to understand how the beta-adrenergic signaling pathway is affected by diabetes, and how these changes may exacerbate and enhance stress on the heart. Activation of cAMP-dependent protein kinase (PKA) via beta-adrenergic receptor signaling is a primary means of increasing cardiac contractility. Over- activation or dysregulation of this pathway is a major driver of diabetic cardiomyopathy, life threatening arrhythmias, and heart failure. However, the mechanisms by which this pathway becomes disrupted are largely unknown. In the healthy heart, PKA increases contractility by amplifying calcium cycling and concertedly activates phosphor-fructose kinase-2 (PFK-2) to promote glucose oxidation. In this manner, workload and metabolic demand are finely orchestrated. The studies of this application address how this orchestration becomes disrupted. The foundation for this work is based on our recent discovery that in the diabetic heart PFK-2 is unresponsive to direct PKA activation, suggesting that post-receptor signaling is compromised. Moreover, our results indicate that this unresponsiveness of PFK-2 is mediated by a decrease in PKA inhibitors (PKIs), a family of nuclear proteins that inhibit PKA and facilitate its transport to the cytoplasm. These findings have led to our overarching hypothesis that diabetes-induces a deficiency of PKI, resulting in aberrant localization and activity of PKA that abrogates PFK2 activation and promotes cardiomyopathy. This hypothesis is being tested and explored using murine models of diabetes and adult primary cardiomyocytes. In Aim 1, we are examining how PKA signaling differs in the diabetic heart and how this changes in cardiac function. Mechanistic studies are being performed to understand how diabetic metabolic conditions mediate these changes. In Aim 2, we are defining the cause and consequence of decreased PKI levels. This aim builds off our recent discovery of the dynamic nature of these proteins. In Aim 3, we are testing the hypothesis that PFK-2 plays an essential role in maintaining metabolic flexibility following beta-adrenergic stimulation. We are defining how PFK-2 levels are affected by diabetes and how this desensitizes this enzyme to PKA activation. These studies will investigate a completely unexplored but critical area of PKA regulation and signaling and how they are impacted by, and perhaps contribute to, diabetic cardiac dysfunction. Results from this study may aid in developing therapeutic targets downstream of beta-receptors.